The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent efflux pump responsible for multidrug resistance in various cancer cell types. This integral plasma membrane protein consists of two homologous halves, each containing an intracellular nucleotide binding domain (NBDl and NBD2). We have characterized the wild-type and mutant MDR1 proteins expressed in a baculovirus-Sf9 system by measuring their drug-stimulated ATPase activity, ATP binding, and vanadate-dependent nucleotide trapping, which reflects a partial reaction of ATP hydrolysis. Kinetic analysis of the full ATP cleavage cycle of the wild-type transporter indicated the presence of more than one functional ATP interacting sites and a positive co-operation between these sites. Mutants carrying a key lysine/methionlne substitution in the NBDl or NBD2 showed significant 8-azido-ATP binding but no ATPase activity or vanadate-dependent nucleotide trapping, independently of the MgATP concentration applied. These results indicate that the steps after the primary binding of MgATP are based on a strong co-operation of the two nucleotide binding domains of MDR1.
|Publication status||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology